The GTPases Rac and Cdc42Hs control diverse cellular functions. In addition to being mediators of intracellular signaling cascades, they have important roles in cell morphogenesis and mitogenesis. A novel PAK related kinase, PAK4, has been identified as a new effector molecule for Cdc42Hs. PAK4 interacts only with the activated form of Cdc42Hs through its GTPase binding domain (GBD). Co-expression of PAK4 and the constitutively active Cdc42HsV12 cause the re-distribution of PAK4 to the Brefeldin A sensitive compartment of the Golgi membrane and the subsequent induction of filopodia and actin polymerization. Importantly, the reorganization of the actin cytoskeleton was dependent on PAK4 kinase activity and on its interaction with Cdc42Hs. Thus, unlike other members of the PAK family, PAK4 provides a novel link between Cdc42Hs and the actin cytoskeleton. The cellular locations of PAK4 and Cdc42Hs suggests a role for the Golgi in cell morphogenesis.
Members of the Rho family of small GTPases Rac and Cdc42Hs have been implicated in diverse biological processes. These include roles in cell proliferation, progression through the cell cycle, and oncogenic transformation (Van Aelst and D'Souza-Schorey, 1997). Cdc42Hs and Rac also play important roles in signal transduction cascades such as those that lead to activation of both the JNK and the p38 families of MAP kinases, and thus lead to long term changes in gene expression (Bagrodia et al., 1995; Brown et al., 1996; Coso et al., 1995; Minden et al., 1995; Zhang et al., 1995). One of the most important functions of Rac and Cdc42Hs is the regulation of the organization of the actin cytoskeleton. Microinjection of Cdc42Hs into fibroblasts and a variety of other cell types causes the induction of filopodia protrusions followed by the formation of lamellipodia. While the induction of filopodia is caused by Cdc42Hs activation, the induction of lamellipodia is probably due to the ability of Cdc42Hs to activate Rac. Thus, co-expression of Cdc42Hs with a dominant negative Rac mutant results in the sustained induction of filopodia without the subsequent induction of lamellipodia. Furthermore, microinjection of activated Rac leads to the induction of lamellipodia, but not filopodia. In addition to the formation of polymerized actin structures, both Cdc42Hs and Rac induce the formation of focal complexes that are associated with the filopodia and lamellipodia. Finally, in some cells, both Cdc42Hs and Rac have been shown to have a role in the dissolution of stress fibers, which may be due to antagonism between these GTPases and a third GTPase, RhoA.
A great deal of effort has been made to identify the downstream molecular targets for Rac and Cdc42Hs. Several proteins were shown to interact with the activated forms of Rac and Cdc42Hs including PAK65, p67-phox, WASP, IQGAP, and MLK3 (Manser et al. 1994, Martin et al. 1995; Bagrodia et al.; 1995, Aspenstrom et al., 1996; Rana et al., 1996; Symons et al., 1996; Hart et al. 1996; Kuroda et al. 1996; Teramoto et al., 1996; Burbelo et al. 1995; Van Aelst et al., 1996). PAK was the first protein kinase that was shown to be a target for Rac and Cdc42Hs, and consequently drew much attention. Activated Rac and Cdc42Hs stimulate PAK autophosphorylation and stimulate its kinase activity. Several PAK family members have been identified and all were shown to interact with GTP bound forms of Rac and Cdc42Hs. These include human PAK1 and 2, mouse PAK3, and the rat homologues PAK .alpha., .beta., and .gamma. (Brown et al., 1996; Manser et al., 1994; Martin et al., 1995; Bagrodia et al.; 1995). The PAKs are all similar in structure, containing an amino terminal regulatory domain and a carboxyl terminal kinase domain. They are also all quite similar in sequence, exhibiting 73% overall sequence identity and approximately 92% sequence identity within the kinase domain (Sells and Chernof, 1997). The regulatory domains of the PAKs contain a GTPase binding domain GBD (Symons et al., 1996) (known also as Cdc42Hs/Rac Interactive Binding (CRIB) domain) (Burbelo et al., 1995) that is necessary and essential for their direct interaction with both Cdc42Hs and Rac.
The functions of the PAKs are not yet entirely known. The sequence similarities between the PAKs and yeast STE20, however, suggests a role in transcription activation or cell morphogenesis. In Saccharomyces cerevisiae, STE20 is activated by Cdc42p, and is an important component of the KSS/FUS3 MAP Kinase pathway. STE20 and the related CLA4 may also mediate cytoskeletal changes induced by Cdc42p, such as those that occur during cytokinesis. Because of the evolutionary conservation between many yeast and mammalian signaling enzymes, it seems likely that the PAKs may have functions similar to the yeast STE20 and CLA4 proteins. In fact, the PAKs have been shown to weakly activate the JNK MAP kinase pathway in some cells (Bagrodia et al., 1995; Brown et al., 1996; Zhang et al., 1995). This suggests that, like STE20, the PAKs may be involved in MAP Kinase pathways. Some groups have shown however, that the PAKs are not necessary for JNK activation, and thus their roles in MAP Kinase pathways are as yet unclear. The PAKs may also be involved in cytoskeletal organization. PAK1 was reported to induce filopodia and membrane ruffles similar to those induced by Cdc42Hs and Rac, and to localize to polymerized actin. Interestingly however, these cytoskeletal changes are partly independent of PAK1's kinase activity, and they also occur independently of PAK1's ability to bind the Rho GTPases. Thus, while overexpressed PAK1 can promote cytoskeletal changes, it may not specifically mediate the cytoskeletal changes induced by Rac and Cdc42Hs. Others have found that PAK1 does not induce filopodia or lamellipodia but instead has a role in the dissolution of stress fibers and down-regulation of focal adhesions. Finally, effector mutants of Rac and Cdc42Hs, (such as RacL61(Y40C) and Cdc42HsL61(Y40C)) that do not bind the PAKs, maintain the ability to induce lamellopodia and filopodia. Taken together, these results suggest that the induction of lamellipodia and filopodia by Rac and Cdc42Hs can occur independently of the known PAKs.
Here the cloning and characterization of a novel serine/threonine kinase, PAK4 is reported. Like other members of the PAK family, PAK4 contains an amino terminal regulatory domain and a carboxyl terminal kinase domain. The kinase domain of PAK4 shares 53% sequence identity with those of the other PAKs. Outside of this region however, PAK4 is entirely different in sequence from the other PAKs, except for a short stretch containing a modified GBD motif. PAK4 is the first member of the PAK family to be identified that differs significantly in sequence from the other PAKs, and thus represents an entirely new member of the PAK family. PAK4 interacts specifically with the GTP bound form of Cdc42Hs via its GBD motif and weakly activates the JNK family of MAP kinases. Co-expression of PAK4 with Cdc42Hs causes PAK4 to translocate from a diffuse perinuclear area to the Golgi membrane and subsequently induced the formation of filopodia and actin polymerization. Thus, the Golgi translocation of PAK4 by Cdc42Hs may be important for its ability to induce filopodia. Furthermore, PAK4 interacts with the Cdc42Hs effector mutant, Cdc42HsL61(Y40C) that was previously was shown to induce filopodia independently of PAKs. These results indicate therefore, that PAK4, rather than the previously identified PAKs, provides a link between Cdc42Hs and the actin cytoskeleton.